Composition and method for optimized hybridization using modified solutions

ABSTRACT

The invention provides a composition, kit and method for hybridizing a probe and target at a temperature lower than their standard hybridization temperature. The chemical component added to the composition has a formula R(NH 2 )C═O, where R is amino or alkyl. A method for use of the chemical component and composition is also disclosed.

FIELD OF THE INVENTION

The present invention relates to the field of nucleic acids and moreparticularly to a modified solution containing a denaturant forapplication with biopolymers and micro arrays.

BACKGROUND OF THE INVENTION

Various arrays of polynucleotides (such as RNA and DNA) are known andused in genetic testing, screening and diagnostics. Arrays are definedby the regions of different biopolymers or nucleotides arranged in apredetermined configuration on a substrate. Most importantly, the arrayswhen exposed to a population of analytes will exhibit a patternindicative of the presence of the various components separatedspatially. Array binding patterns of polynucleotides and/or peptides canbe detected by using a variety of suitable target labels. Once bound tothe array, these target labels can then be quantified and observed andthe overall pattern on the array determined.

DNA micro arrays are particularly useful for analyzing large sets ofgenes through “gene expression profiling”. However, for the micro arraysto be effective in binding target sequences, they must be capable ofannealing. In addition, the detection of optimal “sensitivity” and“specificity” of hybridization for biopolymers (probes) to complimentarysequences in complex cRNA is complicated by the fact that thecharacteristic melting temperature (Tm) for this interaction rises withincreasing probe length. However, increasing the stringency ofhybridization comes with the cost of losing sensitivity. Moreover, thehigher temperatures needed to reach the Tm (>60° C.) are detrimental tothe array surface. Previous researchers have determined that including30% formamide in the hybridization solution can significantly reduce theTm and allow for hybridization to take place at a lower temperaturewhile maintaining an acceptable balance between specificity andsensitivity. Formamide is often used in Southern and Northern blottingas a hybridization modifier, because it is effective and because it iseasily washed out of the nitrocellulose or modified nylon materials usedto perform porous filter-based hybridization assays. However, formamideis a highly toxic and hazardous to ship (the US Department ofTransportation requires double-containment of formamide-containingsolutions). For these reasons it would be desirable to create aneffective solution system that maintains specificity, sensitivity andallows for hybridizations at lower temperature, yet is not toxic, doesnot effect the central nervous system of the operator, is not a fetalpoison or does not require special handling or transportation costs.These and other problems with the prior art systems and solutions areobviated by the present invention. The references cited in thisapplication infra and supra, are hereby incorporated by reference.However, cited references or art are not admitted to be prior art tothis application.

SUMMARY OF THE INVENTION

The invention provides a composition, kit and method for use with microarrays. The composition, kit and method allow a probe and target tohybridize at a temperature lower than their standard hybridizationtemperature. The composition has a chemical component of the formula:R(NH₂)C═Owhere R is an amino or alkyl group. The kit of the present inventioncomprises a micro array, a composition for use with the micro array anda target for detection. The kit may further comprise an optional set ofinstructions. The composition in the kit contains the chemicalcomponent. The chemical component may or may not be in solution. Themethod provides the steps of adding to a probe and target the chemicalcomponent, heating the probe and target in the presence of the addedcomponent and then allowing the biopolymers to hybridize.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to thedrawings in which:

FIG. 1 illustrates a single nucleotide polymorphic array.

FIG. 2 is an enlarged view of a portion of FIG. 1 showing multiple spotsor regions of an array.

FIG. 3 shows a duplex hybridization curve without the addition of thedenaturant. Percentage of single stranded DNA is plotted againsttemperature.

FIG. 4 shows a plot of percentage of single stranded DNA againsttemperature. The plot shows a series of melting curves (25, 60 and 60mer with denaturant added).

FIG. 5 shows a contour plot of the observed optimal conditions usingurea concentrations with varying probe lengths (L=60, T=50° C.). Probelength has been plotted against urea concentration.

FIG. 6 shows a plot of optimal urea concentrations of 50° C. probelength series. The plot shows average GCN4 specificity plotted againsturea concentration.

FIG. 7 shows the extrapolated optimal urea concentration for a probelength of 60-mer at a temperature of 50° C. Optimum urea concentrationat 50° C. is plotted against probe length.

FIG. 8 shows a contour plot of the extrapolation to predicted optimumfor the probe having a length of 60 and 50° C. Probe length has beenplotted against urea concentration.

FIG. 9 shows the results of various arrays with the addition of thedenaturant.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositions,process steps, or equipment, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an array” includes more than one array, reference to “apolynucleotide primer” includes a plurality of polynucleotide primersand the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

A “biopolymer” is a polymer of one or more types of repeating units.This includes traditional polynucleotides, the case in which theconventional backbone has been replaced with a non-naturally occurringor synthetic backbone, and nucleic acids in which one or more of theconventional bases have been replaced with a synthetic base capable ofparticipating in Watson-Crick type hydrogen bonding interactions.Polynucleotides include single or multiple stranded configurations,where one or more of the strands may or may not be completely alignedwith another. While probes and targets of the present invention willtypically be single-stranded, this is not essential. Specifically, a“biopolymer” includes DNA (including cDNA), RNA and polynucleotides,regardless of the source.

An “alkyl” group includes aliphatic and aromatic hydrocarbon moleculesas well as substituted hydrocarbons and their derivatives.

A “nucleotide” refers to a sub-unit of a nucleic acid and has aphosphate group, a 5-carbon sugar and a nitrogen containing base, aswell as analogs of such sub-units. An “oligonucleotide” generally refersto a nucleotide multimer of about 10 to 100 nucleotides in length, whilea “polynucleotide” includes a nucleotide multimer having any number ofnucleotides.

An “array” or “micro array”, unless a contrary intention appears,includes any one or two dimensional arrangement(s) of addressableregions bearing particular biopolymer moieties (for example differentpolynucleotide sequences) associated with that region. An array is“addressable” in that it has multiple regions of different moieties (forexample, different sequences) such that a region at a predeterminedlocation (an “address”) on the array (a “feature” of the array) willdetect a particular target or class of targets (although a feature mayincidentally detect non-targets of the feature). In the present case,the polynucleotide (or other) target will be in a mobile phase(typically fluid), while probes for the target (“probes”) may or may notbe mobile. “Hybridizing”, “annealing” and “binding”, with respect topolynucleotides, are used interchangeably. “Binding efficiency” refersto the productivity of a binding reaction, measured as either theabsolute or relative yield of binding product formed under a given setof conditions in a given amount of time. “Hybridization efficiency” is aparticular sub-class of binding efficiency, and refers to bindingefficiency in the case where the binding components are polynucleotides

The term “Fluid” is used herein to reference to a liquid.

The term “target” shall refer to a nucleic acid, nucleotide, nucleosideor their analogs. The term shall also include nucleotides havingmodified sugars as well as organic and inorganic leaving groups attachedto the purine or pyrimidine rings.

The term “probe” shall refer to a biopolymer such as a nucleic acid,nucleotide, nucleoside or their analogs. The term shall also includenucleotides having modified sugars as well as organic and inorganicleaving groups attached to the purine or pyrimidine rings.

The term “specificity” shall refer to a ratio of specific tonon-specific hybridization.

The term “sensitivity” shall refer to signal intensity of a moleculethat may or may not be attached to a micro array surface.

The term “channel” shall refer to an area on an array that defines aparticular type of feature of the array.

The term “stringent” or “stringency” shall refer to any condition orparameter imposed on a system or hybridization of probe and target thatimproves results (i.e. addition of a denaturant to increasehybridization efficiency).

The term “standard hybridization temperature” shall refer to thetemperature at which two oligonucleotides (i.e. a probe and target)anneal without the addition of any denaturant or other component(s).This is the hybridization temperature of the nucleic acids without anyother components that effect the system thermodynamically. It is alsorelated to a prescribed amount of heat and/or quantum of energy that isadded to a closed system to produce a prescribed level of entropy andWatson-Crick base pairing.

Referring first to FIGS. 1-3, typically kits and methods of the presentinvention use a contiguous substrate 1 carrying arrays 2 disposed acrossan array surface 3 of substrate 1 and separated by interfeature areas 4.The arrays on substrate 1 can be designed for testing an analyte or forevaluating probes on their ability to form hybrids. While a number ofarrays 2 are displayed and shown in FIG. 1, the different embodimentsdescribed below may use substrates with particular numbers of arrays, itwill be understood that substrate 1 and the embodiments to be used withit may use any number of desired arrays 2. For instance, substrate 1 maycarry at least one, two, four, or at least ten arrays 2. Depending uponthe intended use, any or all the arrays 2 may be the same or differentfrom one another and each may contain multiple spots or features 6 ofbiopolymers. A typical array may contain more than ten, more than onehundred, more than one thousand or ten thousand features, or even morethan one hundred thousand features in an area of less than 20 cm² oreven less than 10 cm². For example, features may have widths (that is,diameter, for a round spot) in the range from a 10 μm to 1.0 cm. Inother embodiments each feature 6 may have a width in the range of 1.0 μmto 1.0 mm, usually 5.0 μm to 500 μm, and more usually 10 μm to 200 μm.Non-round features may have area ranges equivalent to that of circularfeatures with the foregoing width (diameter) ranges.

Some or all of the features 6 may be of different compositions. Asmentioned, interfeature areas 4 will typically (but not essentially) bepresent which do not carry any polynucleotide (or other biopolymer of atype of which features 6 are composed). Interfeature areas 4 typicallywill be present where arrays 2 are formed by the conventional in situprocess or by deposition of previously obtained moieties, as describedabove, by depositing for each feature at least one droplet of reagentsuch as from a pulse jet (for example, an inkjet type head). It will beappreciated though, that the interfeature areas 4, when present, couldbe of various sizes and configurations. It will also be appreciated thatthere need not be any space separating arrays 2 from one another. Eachfeature 6 carries a predetermined polynucleotide (which includes thepossibility of mixtures of polynucleotides). As per usual, A, C, G, Trepresent the usual nucleotides. It will be understood that there isusually a linker molecule (not shown) of any known types between thefront surface and the first nucleotide.

FIGS. 1-2 illustrate an ideal array where actual features 6 are the sameas the target (or desired) features, with each feature 6 being uniformin shape, size and composition, and the features 6 being regularlyspaced. Such an array when fabricated by drop deposition methods, wouldrequire all reagent droplets for each feature to be uniform in shape andaccurately deposited at the target feature location. In practice, suchan ideal result is difficult to obtain due to fixed and random errorsduring fabrication. Substrate 1, may have a thickness of less than 1 cm,or even less than 5 mm, 2 mm, 1 mm, or in some embodiments even lessthan 0.5 mm or 0.2 mm. In the situation where the array is read bydetecting fluorescence, substrate 1 may be of a material that emits lowfluorescence upon illumination with the excitation light. The figuresand description should not be interpreted as limiting the scope of theinvention and are for illustrative purposes only. The invention hasapplication to variety of micro array designs that allow biopolymers tohybridize. Similarly, substrate 1 may be of any shape, and any apparatusused with it adapted accordingly.

It should be noted that an important part of the arrays 2 is the abilityof the attached polynucleotides to hybridize to a known or unknowntarget 7 (not shown in FIGS.). Hybridization occurs by annealing ofcomplementary strands. The kinetics of hybridization are well known inthe art and studied. For instance, the kinetics of hybridization for animmobilized target can be defined by:k1[Cf][Cs]where k1 is a constant, [Cf] is the concentration of the bound targetsequence, a constant, [Cs] is the concentration of oligonucleotide insolution. However, a number of experiments have shown that hybridizationdepends on two processes; diffusion of the probe to the site andhybridization at the site. Therefore, the above equation can be writtenand more accurately defined by:k1[Cf][Cs]+Jwhere J is the diffusion coefficient of the probe. From this equation itfollows that when the [Cf] is low, the hybridization is limiting andwhen the concentration of [Cf] is high the diffusion is limiting.

Referring now to FIG. 3, a standard annealing curve is shown. The figureshows a duplex denaturation curve in which percentage of single strandedDNA is plotted against temperature. No denaturant has been added to theDNA. Complementary oligonucleotides are present in solution. The plotshows that as the temperature is increased the probability of annealingis increased. This is probably due to the increased energy that is addedto the oligonucleotide chains in the form of heat. The chains begin torotate and move in solution increasing the probability of complementarystrands and bases to pair and hybridize to form more stable structures.The hybrization temperature is marked by the vertical line in the graphwhere the stability of the duplex formed upon hybridization can beexpressed as a function of the temperature in which 50% of the moleculesare hybridized (Tm).

A molecule such as urea can be used in annealing or hybridizationsreactions to lower the Tm. This is particularly effective in annealingDNA, RNA as a target and especially with dsRNA hybrids, as they havehigh Tms that necessitate elevated reaction temperatures. In a perfectlymatched hybrid, the Tm may depend on a variety of conditions. Someconditions include ionic strength, the concentration and presence orabsence of monovalent cations, base composition of the oligonucleotides,the size of the oligonucleotides, the annealing properties including theamount of mismatches present and the presence of any denaturing agents.For instance, the Tm increases 16.6 degrees Celsius for a one foldincrease in monovalent cations between 0.01 and 0.40 M NaCl. Also, for25 mers, single base deletions show a decrease in Tm by 10 degreesCelsius. For every mismatch the Tm is lowered by 5 degrees Celsius.Denaturing agents such as urea reduce the Tm of a duplex in solution byapproximately 30 degrees Celsius.

FIG. 4 shows a series of melting curves with the addition of urea tocomplementary strands of DNA in solution. The figure shows a plot of %single stranded DNA vs. temperature. Melting curves are shown for 25mer, 60 mer, and 60 mer with denaturant. The solid lines in the diagramindicate the hybridization temperatures after the denaturant urea isadded to the sample. It should be noted that the temperature for optimalduplex formation is generally 5-10 degrees below the Tm. Maximum ratesof hybridization are achieved at temperatures 20-25 degrees Celsiusbelow the Tm (the temperature at which the strands of a double-strandedprobe are half dissociated) (MARMUR, J. G. and DOTY, P. J., Mol. Biol.,3, pp. 584, 1961). Hybridization temperatures at the probe Tm proceedvery slowly. For longer probes, hybridization should be carried out at65-68 degrees Celsius. However, this temperature is detrimental to themicro array surface. Buffer modifiers such as urea significantly reducethe effective Tm for duplex DNA by 15-30 degrees Celsius, enablingannealing to single stranded probes on the micro array surface to occur.This is particularly important for micro array surfaces, where hightemperatures are detrimental to surface stability. A Tm modifier, forexample, 50% formamide, is capable of reducing the Tm of a nucleic acidhybrid so that hybridizations can be carried out at 35-42 degreesCelsius. The advantages of using formamide-based hybridization buffersfor nylon membrane hybridizations is that probes are more stable at thelower temperatures and there is better retention of non-covalently boundnucleic acids on the membrane. Formamide has no effect on hybridizationrate when used in the range of 30-50% (Howley, P. M., et al, J. Biol.Chem., 254, pp. 4876, 1979). The figure clearly shows that as thedenaturant is added to the oligonucleotides in solution, thehybridization temperature is dropped below the original meltingtemperature. This is ideal for use in micro arrays, because less energyis then needed to heat the system to get maximum hybridization.Secondly, since lower heat is applied and the temperature is loweredthere are less denaturing effects on the actual oligonucleotide strands.Also, although the Tm of the 60 mer is higher than the 25 mer, theaddition of denaturant still lowers the Tm and hybridizationtemperature.

The invention provides a solution to be used with the arrays 2. Thesolution contains a chemical moiety having the formula:R(NH₂)C═Owhere R is NH₂ or CH₃. In one embodiment of the invention R is an aminogroup (the resulting compound is urea). In a second embodiment R may bean alkyl group (if R═CH₃, the resulting compound is acetamide). The casewhere R is a hydrogen atom is excluded (when R═H, the resulting compoundis formamide, and the toxicological properties of the compound are verydifferent). Suitable ligands may also include alkyl, substituted alkyl,aryl, substituted aryl, aralkyl, substituted aralkyl, and theirderivatives.

Each of the above embodiments of the invention may be used with asolution that may be added to arrays 2 or other process or apparatusthat allows for the hybridization of a nucleotide, biopolymer, orsimilar type component in a reaction solution or mixture.

Denaturant Buffer Optimization

Tests were conducted in order to achieve hybridization optimization withvarious denaturants. In addition, although short (20-25 base)oligonucleotides should theoretically provide the greatestdiscrimination between related sequences, observation by others haveshown that short oligonucleotide lengths provide poor hybridizationproperties (Guo et al., 1994; Shchepinov et al., 1997). For thesereasons, studies were conducted to maximize hybridization efficiency atlonger lengths such as the 60-mer. These longer oligonucleotides orbiopolymers have the advantage of more closely resembling the solutionstate with enhanced hybridization properties (Guo et al., 1994; Lockhartet al., 1996; Shchepinov et al., 1997).

In order to achieve optimal sensitivity and specificity of hybridizationof 60-mer oligonucleotide arrays to complex target cRNA, a reduction ofthe Tm of the oligonucleotide and target is required. In other words,the length of the oligonucleotide requires added amounts of heat orenergy to obtain hybridizations. However, this added energy or the levelrequired for complete hybridization often requires levels of heat orenergy that can simultaneously destroy the same biopolymers that aredesired to hybridize.

Denaturant Study Structure

Grids experiments were designed using a 3×3×2×2 construction optimizedfor hybridization on the metric of “Mean Specificity” (mean specificityis defined in the equation that follows) as a function of probe length(20 to 60-mer). Tests were conducted using 3, 4, 4.92 M urea at varioustemperatures including 40, 45, and 50° C. Results were duplicated anddye swapped pairs were used to control for any Cy3 vs. Cy5 channel bias(defined as + or − polarity). Mean specificity is given as the averageover all the probes of a given length to a given gene (GCN4=Yel009C).

Samples and Labeling

Samples were labeled with fluorescent dyes (Cy3 and Cy5) forhybridization studies. The specific target used included a pure labeledGCN4 antisense RNA. Cross-hybridization targets included an amplified(polyA+− specific) labeled GCN4/GCN4 yeast knockout (KO) total RNA. Ineach experiment the (+) polarity was the Cy3 labeled specific target andthe Cy5 labeled KO target. The (−) polarity was the Cy5 labeled specifictarget and the Cy3 labeled KO target. Data from each of the polaritieswas combined in the dye swap pairs. Dye swaps were performed toeliminate experimental and channel bias.

Specific Signal vs. Probe Position (GCN4)

Specific signal and probe position are highly correlated. Previously ithas been shown that hybridization efficiency is dependent on probeposition. Probes designed closer to the 3′ end of the corresponding genegive higher signal intensities due to the nature of the enzymetranscriptase activity. In addition, signal strength increases as probelength increases and frequency of good probes increases as probe lengthincreases. These factors were important to consider in the study.

Cross-Hybridization Problem

Cross-hybridization is a problem in hybridization experiments. Increasedhybridization stringency decreases cross-hybridization problems inannealing experiments. Therefore, various parameters must be tested inorder to arrive at maximum sensitivity and specificity with adenaturant. In other words, we want the ideal conditions andconcentrations to produce effective hybridizations below the Tm of abiopolymer and target. The problem with such experiments being thatspecificity (most effective correct binding) to sensitivity (maximumsignal detection) are not easily achieved. In other words, optimalspecificity may be achieved at a different stringency than optimalsensitivity. For formamide, optimal specificity has been observed for ata formamide concentration of approximately 4× that for optimalsensitivity. The KO mutant protocol was employed for comparisons to purecross-hybridization signals. An optimization metric was employed toquantify mean specificity. The mean specificity can be given by theequation: $\quad\begin{matrix}{{+ {Polarity}}\quad} & {\quad{- {Polarity}}}\end{matrix}$${Specificity} = \frac{{\max\left( {{Ssig},{S\quad\min}} \right)}_{g,i} + {\max\left( {{Ssig},{S\quad\min}} \right)}_{r,i}}{{\max\left( {{Xsig},{S\quad\min}} \right)}_{g,i} + {\max\left( {{Xsig},{S\quad\min}} \right)}_{r,i}}$Where,  Ssig  is  SPECIFIC  SIGNAL   Xsig  is  CROSS  HYB  SIGNALSmin = 2.6  σ_(Background)${\overset{N}{Mean}{Specificity}} = {{1/N}{\sum\limits_{i = 1}{Specificity}}}$

Observed Optima for Various Probe Lengths and Temperatures (Urea as aDenaturant)

FIG. 5 shows a contour plot of the observed optimal conditions usingurea concentrations with varying probe lengths. Urea concentrations from2.5 to 5.5 M are plotted on the x-axis and probe lengths were testedfrom 10 to 70-mers using similar targets. The optimum ridge shows theoptimal conditions for hybridization with the addition of the denaturantat 50° C. The ideal is a denaturant concentration that provides thegreatest effect on the varying probe lengths when the temperature isheld constant. The ridge represents this area on the contour plot. Nooptimal urea concentrations were bracket (area that is encircled indiagram) for longer probes at 40 or 45° C. The optimal ureaconcentrations bracketed were at 50° C. for a probe length (L=30, 35, 4045, 50 and 55). No optimal urea concentration was bracketed at 50° C.for L=60. However, the results were extrapolated using the optima forother lengths.

FIG. 6 shows the optimal urea concentrations of 50° C. probe lengthseries. The plot shows average GCN4 specificity plotted against ureaconcentration. Each of the curves is concave down with a maxima around4.0 M urea concentration. The curves in each case are parabolic and showpredictable trends.

FIG. 7 shows the extrapolated optimal urea concentration for a probelength of 60-mer at a temperature of 50° C. Optimum urea concentrationat 50° C. is plotted against probe length. Probe length is graphed from25 to 65-mer. For the 60-mer the optimal urea concentration is predictedto be between 5.1 and 5.3 M.

FIG. 8 shows a contour plot of the extrapolation to predicted optimumfor the probe having a length of 60. Probe length has been plottedagainst urea concentration. The ridge in the diagram shows the optimumpredicted for the denaturant on the prescribed conditions. The diagramshows that the optima for these experiments are very broad. Therefore,denaturants added to a probe and target can be very effective inlowering Tm for improved hybridizations. Maximum urea concentrationbeing approximately 4.92 M.

FIG. 9 shows the results of the Cy3 and Cy5 labeled micro arrays. Thediagram shows the effects of oligonucleotide length and hybridizationstringency on hybridization to yeast genes. Three yeast genes are shown:YDR345C (HXTT3), YER0109W (ISC1) and (GCN4) are tiled on a DNAoligonucleotide micro array. The oligonucleotide probes corresponding toeach of the three yeast cDNAs are of increasing length, 20, 25, 30, 35,40, 45, 50, 55, 60-mer, and have been tilted along the gene, spacedthree oligonucleotides apart and arrayed in a 75 rows×195 column format.Reverse gray images reveal hybridization of oligonucleotides probes in4.92 M urea at 40° C. Cy4 labeled Yeast GCN4 KO cRNA (2 μg), highlitesHXT3 probes on the array. Whereas PCR amplified Cy3 labeled full lengthGCN4 DNA (0.5 ng) highlites GCN4 probes. Probes hybridizing equally toCy5 and Cy3 are labeled yellow. ISC1, in the middle of the array, is ano-expressing gene. The diagrams clearly show that hybridization takesplace at lower temperature with the added denaturant. Similarhybridizations can be seen in the low and high temperature micro arrays.However, the higher temperatures show slight problems with delamination,while the lower temperature graphs show an absence of such problems. Foran example of the solution(s) used in the experiments see Tables 1-3.Table 1 is the general buffer system. Table 2 shows a solution to beemployed in the Table 1 preparation.

The invention also provides a kit for using the composition. The kitprovides for hybridizing biopolymers at temperatures below theirstandard hybridization temperatures. The kit comprises a micro array; acomposition for use with the micro arrays, a target for detection and anoptional set of instructions. The optional set of instructions maydescribe how to use the denaturant to obtain most effective bindinghybridization temperatures. The micro array may be constructed of anynumber of probe lengths. The target may be a known or unknownbiopolymer. The composition includes the chemical component R(NH₂)C═Owhere R is an amino or an alkyl group. The chemical component may beused alone or in solution.

Having described the composition and kit of the present invention, abrief description of the method is now in order. The method includes theapplication or use of the same chemical composition. The composition mayor may not be in a buffered solution. The method allows a probe tohybridize to a target at a temperature lower than their standardhybridization temperature. The steps of the method comprise adding tothe probe and target a chemical component of the formula: R(NH₂)C═Owhere R is an amino or an alkyl group, heating the probe and target inthe presence of the added component, and then allowing the probe andtarget to hybridize.

EXAMPLE 1 Micro Arrays

Spotted cDNA arrays. Robotically deposited arrays of PCR products, eachcorresponding to a single yeast ORF, were as previously described(Marton, M. J. et al., 1998).

Oligonucleotide arrays. Surface-bound oligonucleotides were synthesizedessentially as proposed (Blanchard et al., 1996). Hydrophobic glasswafers (75×75 mm) containing exposed hydroxyl groups for nucleotidecoupling were prepared using a self assembling silane mixture to form auniform, hydrophobic surface with a controlled density of hydroxylgroups extending by a single methylene unit above unfunctionalizedsilane components (S. M. Lefkowitz et al, unpublished data).Phosphoramidite monomers in propylene carbonate were delivered todefined positions on the glass surfaces using commercial ink-jet printerheads. Synthesis cycles were otherwise similar to traditionaloligonucleotide synthesis, with the exception that capping was omitted.After manufacture, arrays were diced to 25×75 mm to accommodatecommercial scanners.

EXAMPLE 2 Oligonucleotide Selection for Micro Arrays

Saccharomyces cerevisiae oligonucleotide set design were constructed asdescribed below. Sequences were chosen from the full genome sequence(obtained from http://genome-www.stanford.edu/Saccharomyces). All 60-mersequences with the 3′ kb of nuclear encoded ORF sequences wereevaluated. First, low-complexity sequence was masked using RepeatMasker(University of Washington and Genetic Information Research Institute).Second probes with 35-45% (G+C) were taken. Among these, a set ofoligonucleotides overlapping by <40 bases was derived and analyzed byBLAST (http://www.ncbi.nlm.nih.gov/BLAST/) to identify those with lowestsimilarity to other source sequences. The eight remaining probes withthe highest predicted specificity were selected to represent the sourcesequence. In some cases, eight oligonucleotides meeting these criteriawere not identified, in which case selection stringencies were loweredand/or fewer than eight oligonucleotides were printed.

Human oligonucleotide sets were designed as described below. A total of49,218 Homo sapiens sequences were picked from the longest messenger RNA(mRNA) sequences representing UniGene clusters (Release 111, 15 Apr.,1999) (http://www/ncbi.nlm.nih.gov/UniGene/). For each sequence, every60-mer between 50 and 350 bases from the 3′ end of the sequence wasevaluated. First, oligonucleotides were filtered to remove with anyrepeat elements. Simple repeats or repeat elements were detected usingRepeat Masker. Oligonucleotides with homopolymeric stretches of morethan six bases were also rejected. Second, oligonucleotides werefiltered to be <50% G and 25-70% (G+C). Among those remaining, a set ofoligonucleotides overlapping by <30 bases was derived and analyzed byBLAST to identify those with lowest similarity to all other sourcesequences. The surviving oligonucleotide with the highest predictedspecificity was selected to represent its source sequence. The 23,965oligonucleotides on the array used are those among 49,218 with thehighest signal intensity when hybridized with cRNA prepared from Jurkatcells.

Mutations and deletions were performed as described below. Mismatchbases were “mutated” at random, for example, a mutated guanine had anequal possibility of mutation to each of the other possible bases(adenine, cytosine, or thymidine). In the case of deletions, additionalsequence adjacent on the synthetic mRNA was added so that a total of 60complementary bases was retained. For each number of randomly placedsingle-base mismatches or deletions, 110 different oligonucleotides weredesigned. For sequential mismatches, each position was represented by asingle oligonucleotide sequence printed on five different spots.

EXAMPLE 3 Cell Culture

Saccharomyces cerevisiae cultures were grown as described (Marton et.al., 1998). The gcn4Δ/gcn4Δ strain is R1792; the hxt3Δ/hxt3Δ strain isR5918.Strains that are often used and profiled are prototrophic diploid(i.e. R1165). NB4 cells (human promyelocytic leukemia) were obtainedfrom the American Type Culture Collection (Rockville, Md.). Human celllines were grown in RPMI medium supplemented with 10% fetal bovine serumin an atmosphere of 5% CO₂.

EXAMPLE 4 RNA Isolation

Yeast total RNA was isolated by phenol:chloroform extraction and poly-Apurified as described (Marton et al., 1998). Human total RNA wasisolated using TRIzol (Life Technologies, Rockville, Md.) or Rneasy(Qiagen, Valencia, Calif.).

EXAMPLE 5 Synthetic cRNA and mRNAs

Full length S. cerevisiae ORFs GCN4 and HXT3 were amplified by PCR froma genomic DNA template and ORF specific primers. The T7 RNA polymerase(T7RNAP) promoter sequence was introduced into the antisense primer foreach ORF. ORF specific cRNAs were then produced from the DNA templatesby in vitro transcription using a kit well know in the art (MegaScript,Ambion, Austin Tex.). After labeling (as described below), thesesynthetic cRNAs were used directly for micro array hybridization. Thearray was hybridized with a mixture of 2 μg Cy3-labeled cRNA from a GCN4deficient strain of S. cerevisiae and 0.5 ng Cy5-labeled GCN4ORF-specific IVT product (cRNA) (˜4.5 copies per cell of the 843 ntGCN4, ORF, assuming ˜15,000 transcripts per S. cerevisiae and an averagetranscript length of 1 kb). For spike-in RNAs, a portion of theadenovirus type 5 Ela (nucleotides 560-972) was amplified by PCR from afull-length plasmid clone of the gene and subcloned into the pS64Poly(A)Vector (Promega, Madison, Wis.). Random 60-mer oligonucleotides werethen cloned into Xbai/BamHI sites of this subclone, adjacent the poly-Asequence. Individual clones were isolated, and the sequences of theinserts determined. To generated synthetic mRNAs, clones were linearizedwith EcoRI and an SP6 IVT reaction was then performed. This reaction wasthen followed by DNase treatment of the product. Synthetic mRNAs werepurified on Rneasy columns (Qiagen).

EXAMPLE 6 MRNA Reverse Transcription and Amplification

In vitro transcription (IVT) was performed as modified (DeRisi, et al.,1997). Total RNA was used as input for cRNA synthesis. An oligo-dTprimer containing a T7 RNA polymerase promoter sequence was used toprime first strand cDNA synthesis, and random hexamer were used to primesecond stranded cDNA synthesis by Maloney murine leukemia virus(MMLV)RT. This reaction yielded a double stranded cDNA that containedthe T7 RNA polymerase promoter at the 3′ end. The double stranded cDNAwas then transcribed into cRNA by T7RNAP.

PCR-coupled IVT (PCR-IVT) was performed as described below. 3′end cDNAwas synthesized by an adaptation of the protocol of Zhao et al. (Zhao etal., 1998). To prevent transcript detection biases due to unequalamplification of certain sequences during PCR, the amount of input RNAwas increased to 2.4-4.0 μg and decreased the number of PCR cycles to10. To allow further sequence amplification by cRNA synthesis, a T7RNAPpromoter sequence was added to the 3′-end primer sequence used duringPCR. Following PCR, amplified DNA was isolated by phenol/chloroformextraction and used as a template in an IVT reaction (MegaScriptAmbion).

EXAMPLE 7 Sample Labeling, Hybridization, Scanning and Image Analysis

Different cDNA and cRNA were labeled with Cy3 and Cy5 dyes using a twostep process. First, allylamine-derivitized nucleotides wereenzymatically incorporated into cDNA or cRNA products. For cDNAlabeling, a 1:1 mixture of 5-(3-aminoallyl) thymidine 5′-triphosphate(Sigma) and thymidine triphosphate (TTP) was used in place of TTP duringcDNA synthesis. For cRNA labeling, a 1:3 mixture of5-(3-aminoallyl)uridine5′-triphosphate (UTP) was substituted for UTP inthe IVT reaction. Allylamine derivatized cRNA or c DNA products werepurified using commercial columns. Columns were washed 3× with 80%ethanol and eluted with water. Purified products were reacted withN-hydroxy succinimide esters of Cy3 or Cy5, following the manufacturer'sinstructions. Dye molecules were separated from labeled products usingstandard commercial columns. Cy3 labeled cRNA or cDNA from “control”cells were mixed with the same amount of Cy5-labeled product from“experiment” cells (˜30 μg for cDNA or 5 μg for cRNA per human sampleper hybridization, unless otherwise noted). All hybridizations were donein duplicate and fluor reversal, to compensate for biases cause bydiffering chemical properties of the fluorescent dye molecules as wellas for biases associated with normalization. Before hybridization,labeled cRNAs were fragmented to an average size of ˜50-100 nt beforeincubation at 60, 50, 45, or 40 degrees Celsius for 30 minutes in thepresence of 10 mM ZnCl₂, cDNAs or fragmented cRNAs were added tohybridization buffer as described below. Hybridizations were carried outin a final volume of 3 ml in a flexible plastic enclosure at 40 degreesCelsius on a rotating platform in a hybridization oven (RobbinsScientific, Sunnyvale, Calif.). After hybridization for 16-24 hours,slides were washed (rocking ˜30 s in 6×SSPE, 0.005% sarcosine, thenrocking 30 s in 0.06×SSPE) and scanned using a confocal laser scanner(General Scanning, Wilmington, Mass.; Genetic Microsystems, Woburn,Mass.; or Axon, Foster City, Calif.). Fluorescence intensities onscanned images were quantified, corrected for background and normalized(Marton et al., 1998).

EXAMPLE 8 The Solution or Reaction Mixture

Table 1-3 shows a description and breakdown of the components used in areaction solution. The components generally include nuclease free water,5 M MCI (M=Na⁺, Li⁺, or other suitable monovalent cation), 1.0 M M-MES,pH=6.4, and 10% w/v Sarcosine (sigma L-9150). The volumes for each ofthe components are shown in the tables for the case where M=Na⁺.Solutions may be altered and components changed. The guiding designprinciple is that the addition of the urea, amide, or an amino componentallows for hybridization of the polynucleotide or biopolymer on themicro array surface at lower temperature than the standard hybridizationtemperature without the addition of the denaturant. The optimization ofthe chosen temperature and the composition of the hybridization bufferrepresents a balancing of the stabilization of perfect Watson-Cricknucleic acid duplexes and the destabilization of imperfect duplexes,i.e. the balance between sensitivity and specificity. [NOTE: optimalhybridization is usually achieved at a temperature about 5° C., belowthe melting temperature of the desired duplex. Melting temperature iseffected by monovalent cations (usually Na⁺ or Li⁺) concentration, probelength, probe sequence, modifier concentration and target concentration.The affect of the modifier (urea, acetamide, etc.) is to lower themelting temperature, all other factors being equal, by destabilizing thehydrogen bonding of Watson-Crick base pairs. -pKw].

EXAMPLE 9 Effects of Various Molarities on Oligonucleotide HybridizationTemperature

FIGS. 5-9 show the effects of various urea molarities on thepolynucleotide or biopolymer hybridization temperature. As can be seenin the figures, higher amounts of urea are effective in loweringhybridization temperatures. Note that as the probe length increases,there is also a higher required molarity of urea to obtain the maximumor most effective hybridization temperature.

For further questions regarding any of the procedures in the describedexamples see (Hughes et al., 2001). TABLE 1 Hybridization Title: GCN4Experiment Protocol Dye swap Polarity (—) Array Design FileScerevisiae_hxt3_yer019w_gcn4_20-60mers.txt Total Number ofslides/Temp/Treat 2 Final Volume per slide: 3000.0 μl 4.92M Urea[Component] Volume Amount Final [Component]

FRAGMENTATION STEP nuclease-free water 100.0% 110.0 μl 3.7% GCN4 spike(S. Cerevisiae) Cy5 0.10 μg/ml 10.20000 0.5 ng 0.00017 μg/ml gcn4 KO (S.Cerevisiae) Cy3 339.30 μg/ml 11.85 μl 2 μg 0.67 μg/ml Random 25-mer10.00 mg/ml 60.00 μl 0.10 mg/ml 25 mM Zn(OAc)₂, 975 mM Tris, pH 7.6 25x8.00 μl 1.0x Fragmentation volume: 100.00 μl 200.0 μl 0.5M EDTAPOST-fragmentation stop volume 5.00 μl 10.00 μl Total Fragmented Volume:210.00 μl HYBRIDIZATION STEP Fragmented sample volume per tube: 210.0 μlUrea Volume Concentrate (8M Urea) 3690.0 μl 4.92M Urea 20/7X Hyb BufferConcentrate 2100.0 μl 1X Water 0.0 μl Total 6000.0 μl

TABLE 2 Desired Volume (ml): 25 20/7x (2.86x ) Urea Hyb. Buffer Final2.86x [Component]/2.86x Component [Component] Volume (ml) Concentrate(in hyb. chamber) nuclease-free water 100.0% 2.71 10.9% 3.8% 5M Nacl(nuclease-free) 5.0 M 14.29 2.9 M 1.0 M 1.0M Na-MES, pH 6.4 1000.0 mM3.57 142.9 mM 50.0 mM 0.5 M EDTA (pH 8.0) 500.0 mM 0.86 17.1 mM 6.0 mM10% w/v Sarcosine (Sigma L-9150)  10.0% 3.57  1.4% 0.5% Total 25.0 ml

TABLE 3 Desired Volume (ml): 10 2X QC2-3 Hyb. Buffer Final[Component]/2X Volume 2X (in hyb. Component [Component] (ml) Concentratechamber) nuclease-free water 100.0% 0.23 2.3% 1.15% 8M LiCl(nuclease-free) 8.0 M 1.53 1.224 M 0.612 M 0.5M Li-MES, pH 6.1 500.0 mM6.00 300.0 mM 150.0 mM 0.5 M EDTA (pH 8.0) 500.0 mM 0.24 12.0 mM 6.0 mM15% w/v Lithium Dodecyl Sulfate, 10% Triton X-100  15.0% 2.00 3.0% 1.5% Total 10.0 ml

1-17. (canceled)
 18. A composition of matter comprising: a) asurface-bound oligonucleotide probe; b) a polynucleotide target for saidprobe; and c) urea.
 19. The composition of matter of claim 18, whereinsaid surface-bound oligonucleotide probe is attached to a surface of asubstrate.
 20. The composition of matter of claim 19, wherein saidsubstrate is a glass substrate.
 21. The composition of matter of claim19, wherein said substrate comprises an array of at least 1000surface-bound oligonucleotide probes.
 22. The composition of matter ofclaim 18, wherein said surface-bound oligonucleotide probe is 10 to 100nucleotides in length.
 23. The composition of matter of claim 18,wherein said urea is present in an amount that decreases the standardhybridization temperature of said oligonucleotide probe and saidpolynucleotide target.
 24. The composition of matter of claim 18,wherein said hybridization reagent is at a temperature in the range of35° C. to 42° C.
 25. The composition of matter of claim 18, wherein saidurea is present at a concentration of 2.5 M to 5.5 M.
 26. A kitcomprising: an array of surface-bound oligonucleotide probes; and ahybridization buffer comprising urea.
 27. The kit of claim 26, whereinsaid surface-bound oligonucleotide probes are linked to a glasssubstrate.
 28. The kit of claim 26, wherein said array is an array of atleast 1000 surface-bound oligonucleotide probes.
 29. The kit of claim26, wherein said surface-bound oligonucleotide probes are 10 to 100nucleotides in length.
 30. The kit of claim 26, wherein said urea ispresent at a concentration of 2.5 M to 5.5 M.
 31. A composition ofmatter comprising: a) a surface-bound oligonucleotide probe; b) apolynucleotide target for said probe; and c) acetamide.
 32. Thecomposition of matter of claim 31, wherein said surface-boundoligonucleotide probe is attached to a surface of a substrate.
 33. Thecomposition of matter of claim 32, wherein said substrate is a glasssubstrate.
 34. A kit comprising: an array of surface-boundoligonucleotide probes; and a hybridization buffer comprising acetamide.35. The kit of claim 34, wherein said surface-bound oligonucleotideprobes are linked to a glass substrate.
 36. The kit of claim 34, whereinsaid array is an array of at least 1000 surface-bound oligonucleotideprobes.